In vitro growth culture of bovine follicles for developing model of dominant follicle and for application to in vitro production of wildlife oocytes

• Project/Area Number: 25450441
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Veterinary medical science
• Research Institution: Hokkaido University
• Principal Investigator: Nagano Masashi 北海道大学, (連合)獣医学研究科, 准教授 (70312402)
• Research Collaborator: HUANG Weiping ; KANG Sung-Sik ; KOYAMA Keisuke ; NAGAI Katsuhisa ; SAKAGUCHI Kenichiro ; YANG Yinghua
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
• Keywords: 牛 / 卵子 / 体外発育 / 体外受精 / 卵胞 / 体外発育培養 / 野生動物 / 精子 / IVG / IVM / 初期胞状卵胞 / 受精卵移植

Outline of Final Research Achievements

I tried to improve the culture system of oocyte-granulosa complexes (OGCs) derived from bovine early antral follicles. As the results, I produced offspring effectively by my developed system, and it was the third case in the world. The result indicates the embryos derived from the culture system have normality the same as in vivo derived embryos. When I added born morphogenetic protein-4 (BMP-4) to culture medium, the mean diameter of granulosa cells became smaller and progesterone secretion from granulosa cells was inhibited. These mean that BMP-4 inhibits the luteinization of granulosa cells. In addition, I compared the developmental competence of OGCs derived from ovaries having different antral follicle count (AFC). OGCs derived from high and low AFCs showed similar viability during growth culture; however, the mean number of granulosa cells was higher in high AFC group. Oocytes derived from high AFC have higher activity of mitochondria and showed higher nuclear maturation rate.

Research Products

• [Journal Article] Mitochondrial activity during pre-maturational culture in in vitro-grown bovine oocytes related to maturational and developmental competences (2016)
  • Author(s): Huang, W., Kang, S.S., Nagai, K., Yanagawa, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Reproduction, Fertility and Development Volume: 28 Issue: 3 Pages: 349-356
  • DOI: 10.1071/rd14023
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] <b>The relationship between antral follicle count in a bovine ovary and developmental competence of </b><i><b>in vitro</b></i><b>-grown oocytes derived from early antral follicle</b><b>s </b> (2016)
  • Author(s): Nagai, K., Yanagawa, Y., Katagiri, S. and Nagano, M.
  • Journal Title: Biomedical Research Volume: 37 Issue: 1 Pages: 63-71
  • DOI: 10.2220/biomedres.37.63
  • NAID: 130005128378
  • ISSN: 0388-6107, 1880-313X
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Effect of bone morphogenetic protein-4 on in vitro growth, steroidogenesis and subsequent developmental competence of the oocyte-granulosa cell complex derived from bovine early antral follicles (2016)
  • Author(s): Yang Y, Kanno C, Huang W, Kang SS, Yanagawa Y, Nagano M.
  • Journal Title: Reproductive Biology and Endocrinology Volume: 14 Pages: 3-3
  • NAID: 120005725145
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flowcytometry (2016)
  • Author(s): Kanno, C., Kang, S.S., Kitade, Y., Yanagawa, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Zygote Volume: 未定 Issue: 4 Pages: 529-536
  • DOI: 10.1017/s0967199415000490
  • NAID: 120005740328
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Addition of D-penicillamine, hypotaurine, and epinephrine (PHE) mixture to IVF medium maintains motility and longevity of bovine sperm and enhances stable production of blastocysts <i>in vitro</i> (2015)
  • Author(s): Kang, S.S., Koyama, K., Huang, W., Yanagawa, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Journal of Reproduction and Development Volume: 61 Issue: 2 Pages: 99-105
  • DOI: 10.1262/jrd.2014-112
  • NAID: 130005064911
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Fertilizability of oocytes derived from Holstein cows having different antral follicle counts in ovaries (2015)
  • Author(s): Nagai, K., Yanagawa, Y., Katagiri, S. and Nagano, M.
  • Journal Title: Animal Reproduction Science Volume: 163 Pages: 172-178
  • DOI: 10.1016/j.anireprosci.2015.11.009
  • NAID: 120005890013
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Effects of in vitro-growth culture duration on fertilizability of bovine growing oocytes and proliferation of cells surrounding oocytes (2014)
  • Author(s): Huang, W., Kang, S.S., Yanagawa, Y., Yang, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Japanese Journal of Veterinary Research Volume: 62 Issue: 3 Pages: 135-141
  • DOI: 10.14943/jjvr.62.3.135
  • NAID: 120005469242
  • ISSN: 0047-1917
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Aging-related Changes in <i>In Vitro</i>-matured Bovine Oocytes: Oxidative Stress, Mitochondrial Activity and ATP Content After Nuclear Maturation (2014)
  • Author(s): Koyama, K., Kang, S.S., Huang, W., Yanagawa, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Journal of Reproduction and Development Volume: 60 Issue: 2 Pages: 136-142
  • DOI: 10.1262/jrd.2013-115
  • NAID: 130003390821
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Estimation of the Optimal Timing of Fertilization for Embryo Development of <i>In Vitro</i>-Matured Bovine Oocytes Based on the Times of Nuclear Maturation and Sperm Penetration (2014)
  • Author(s): Koyama, K., Kang, S.S., Huang, W., Yanagawa, Y., Takahashi, Y. and Nagano, M.
  • Journal Title: Journal of Veterinary Medical Science Volume: 76 Issue: 5 Pages: 653-659
  • DOI: 10.1292/jvms.13-0607
  • NAID: 130003391268
  • ISSN: 0916-7250, 1347-7439
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Prematurational Culture with 3-Isobutyl-1-methylxanthine Synchronizes Meiotic Progression of the Germinal Vesicle Stage and Improves Nuclear Maturation and Embryonic Development in <i>In Vitro</i>-grown Bovine Oocytes (2014)
  • Author(s): Huang, W., Nagano, M., Kang, S.S., Yanagawa, Y. and Takahashi, Y.
  • Journal Title: Journal of Reproduction and Development Volume: 60 Issue: 1 Pages: 9-13
  • DOI: 10.1262/jrd.2013-082
  • NAID: 130003381855
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed
• [Journal Article] Effects of in vitro growth culture duration and pre-maturation culture on maturational and developmental competences of bovine oocytes derived from early antral follicles (2013)
  • Author(s): Huang, W., Nagano, M., Kang, S.S., Yanagawa, Y. and Takahashi, Y.
  • Journal Title: Theriogenology Volume: 80 Issue: 7 Pages: 793-799
  • DOI: 10.1016/j.theriogenology.2013.07.004
  • NAID: 120005345325
  • Peer Reviewed
• [Journal Article] In vitro maturation system for individual culture of bovine oocytes using micro-volume multi-well plate (2013)
  • Author(s): Nagano, M., Kang, S.S., Koyama, K., Huang, W., Yanagawa, Y. and Takahashi, Y.
  • Journal Title: Japanese Journal of Veterinary Research Volume: 61 Pages: 149-154
  • NAID: 120005350105
  • Peer Reviewed
• [Presentation] 牛の卵子品質と卵巣内卵胞数の関係: 体内および体外発育卵子を用いた予備的検討 (2015)
  • Author(s): 永井克尚，杉山ちさと，楊応華，栁川洋二郎, 片桐成二，永野昌志
  • Organizer: 第108回日本繁殖生物学会大会
  • Place of Presentation: 宮崎県宮崎市学園木花台西1-1 宮崎大学木花キャンパス
  • Year and Date: 2015-09-17
• [Presentation] Effects of bone morphogenetic protein-4 (BMP-4) on the acquisition of developmental competence of oocytes derived from bovine early antral follicles (2015)
  • Author(s): Yinghua Yang, Sung-Sik Kang, Yojiro Yanagawa, Masashi Nagano
  • Organizer: the fifth meeting of the Ovarian Club
  • Place of Presentation: 香港
  • Year and Date: 2015-01-31 – 2015-02-01
• [Presentation] ニホンザル（Macaca fuscata）の周排卵期における卵巣およびホルモン動態 (2014)
  • Author(s): 南晶子、栁川洋二郎、印藤頼子、兼子明久、岡本宗裕、永野昌志
  • Organizer: 第20回日本野生動物医学会つくば大会
  • Place of Presentation: つくば市
  • Year and Date: 2014-09-16 – 2014-09-19
• [Presentation] ヒグマ（Urusus arctos）におけるカテーテル法を用いた精液採取：オキシトシン投与が精液採取と精子品質に与える影響 (2014)
  • Author(s): 栁川洋二郎、簑原悠太朗、冨安洵平、松本直也、坂本秀行、松井基純、永野昌志
  • Organizer: 第20回日本野生動物医学会つくば大会
  • Place of Presentation: つくば市
  • Year and Date: 2014-09-16 – 2014-09-19
• [Presentation] カテーテル法を用いたホッキョクグマ（Ursus maritimus）の採精と造精機能向上への取り組み (2014)
  • Author(s): 中村亮平、佐藤伸高、佐橋智弘、坂東 元、栁川洋二郎、松井基純、楠田哲士、永野昌志
  • Organizer: 第20回日本野生動物医学会つくば大会
  • Place of Presentation: つくば市
  • Year and Date: 2014-09-16 – 2014-09-19
• [Presentation] he effects of PHE mixture, theophylline, and sperm concentrations on fertilization and development of bovine oocytes in vitro (2014)
  • Author(s): Sung-Sik Kang, Yojiro Yanagawa, Masashi Nagano
  • Organizer: World Congress of Reproductive Biology 2014
  • Place of Presentation: エジンバラ（スコットランド）
  • Year and Date: 2014-09-02 – 2014-09-04
• [Presentation] The sperm penetration rate and embryo development kinetics of embryos fertilized with sex-sorted sperm in bovine (2014)
  • Author(s): Sung-Sik Kang, Yoshiyuki Takahashi, Yojiro Yanagawa, Masashi Nagano
  • Organizer: The Korean Society of Embryo Transfer 2014
  • Place of Presentation: 大韓民国
  • Year and Date: 2014-06-05 – 2014-06-06
• [Presentation] Pre-IVM culture with IBMX improves maturational and developmental competences of bovine oocytes derived from in-vitro growth culture (2013)
  • Author(s): Weiping HUANG, Masashi NAGANO, Sung-Sik KANG, Yojiro YANAGAWA, Yoshiyuki TAKAHASHI
  • Organizer: 第106回日本繁殖生物学会大会
  • Place of Presentation: 東京農工大学（東京都）
• [Presentation] 牛初期胞状卵胞由来卵子の体外発育に及ぼすFSHと骨形成タンパク質4の影響 (2013)
  • Author(s): 坂口謙一郎、栁川洋二郎、姜成植、黄偉平、髙橋芳幸、永野昌志
  • Organizer: 第106回日本繁殖生物学会大会
  • Place of Presentation: 東京農工大学（東京都）
• [Presentation] Effects of in-vitro growth (IVG) duration and pre-maturational culture on maturational and developmental competences of oocytes derived from early antral follicles (2013)
  • Author(s): Weiping HUANG, Masashi NAGANO, Sung-Sik KANG, Yojiro YANAGAWA, Yoshiyuki TAKAHASHI
  • Organizer: 第156回日本獣医学会学術集会
  • Place of Presentation: 岐阜大学（岐阜県）
• [Remarks] 北海道大学大学院獣学研究科診断治療学講座繁殖学教室
  • URL: http://www.vetmed.hokudai.ac.jp/organization/theriogenol/index.html